This component will continue elucidating the molecular mechanisms underlying ontogenesis of kappa opioid receptor (KOR), i.e. production of KOR protein during developmental stages and differentiating neurons. Studies in the previous funding cycles have delineated several regulatory pathways for the control of KOR mRNA production during developmental stages, principally transcriptional control. This concludes the first phase of studies focusing on the regulation of KOR mRNA synthesis, a hall markd of KOR neurogenesis involving signaling pathways of retinoic acid (vitamin A) and nitric oxide and requires chromatin remodeling of KOR gene regulatory regions, as well as more recent findings in epigenetic control. Importantly, ontogenesis of KOR appears to be controlled, most crucially, by post-transcriptional mechanisms such as mRNA stability, transport and translation. This renewal component will focus on translational mechanism by extending from our preliminary studies that have identified Netrin-1 as a translational stimulator for KOR, and Grb7 as a translational represser of KOR. Two specfiic aims are: 1. To elucidate the mechanism that activates KOR translation via Netrin-1/Grb7 pathway. We will focus on i) regulation of translational initiation of KOR by Grb7, including studies of its molecular and biochemical features and funcitonal domains, and KOR RNA sequences bound by Grb7 which can be activated by Netrin-1, and ii) specific translational initiation step that is targeted by Grb7 including initiation factors and subcellular distribution and possible circularization of KOR mRNA. 2. Pharmacological and physiological relevance of Grb7/Netrin-1 signaling to KOR ontogenesis. We will i) perform gain- and loss-of-function studies to validate the relevance of Netrin-1/Grb7 in KOR ontogenesis using stem cells and primary neurons, and ii) examine the physiological relevance of KOR synthesis in neuronal activity such as in a specific pain circiitry and during nerve injury or as a stress response.